April 2009 technical highlight
Improve your cloning efficiency
New vectors and a step-by-step protocol will make ligation-independent cloning even simpler.
Many researchers swear by ligation-independent cloning, finding it faster and more efficient than conventional methods. PSI MCSG routinely achieves efficiencies of between 75% and 90% using ligation-independent cloning, and now they take this technique a step further, producing specialized vectors for coexpression and co-purification of interacting proteins.
Several ligation-independent strategies exist: PSI JCSG uses a polymerase incomplete primer extension (PIPE) approach, whereas PSI MCSG uses a T4 polymerase method. The latter technique uses the exonuclease activity of T4 polymerase to generate long overhanging ends of 12 to 15 nucleotides in the vector and the insert rather than the short overhang produced by restriction enzyme digestion. The longer stretch of nucleotides is sufficient for insert binding after introducing the vector and its insert into a host bacterium adding a ligase to the in vitro mixture is not necessary.
In Methods in Molecular Biology, Eschenfeldt et al. from PSI MSCG describe a family of 15 ligation-independent vectors they use to overcome common protein production problems and provide a detailed protocol for manual insertion of genes. The cloning process is identical for all the vectors and, briefly, consists of linearizing the vector by treating it with the endonuclease SspI, followed by T4 polymerase treatment in the presence of dGTP. The exonuclease activity of the polymerase hydrolyzes nucleotides from the 3' ends of the vector until it reaches a guanine reside, thus creating a 15-nucleotide overhang. The DNA to be inserted is similarly treated with the polymerase in the presence of dCTP only to create a complementary overhang. The two DNA fragments are then mixed, allowed to anneal and introduced into a host cell to complete the ligation.
The 15 vectors from PSI MCSG, including vectors for decreasing protein toxicity, increasing solubility and for enabling coexpression of two proteins, are available through the PSI Materials Repository. This is a non-profit service aimed at sharing the PSI's resources with a wider community.